# Dynamic allosteric communication within nonribosomal peptide synthetase cyclization domains

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2021 · $104,280

## Abstract

Project Summary
Biological activity, ranging from gene activation to enzyme regulation, occurs through molecular
interactions, and its regulation can be described as a redistribution of intermolecular interactions
through chemical modifications or ligand binding. Unfortunately, when a protein interacts with
two partners through remote binding sites, molecular mechanisms that would explain how
changes within proteins alter the communication between proteins are often elusive. This
challenge limits designing drugs that could alter interactions to rescue abnormal biological
activity. The conundrum also applies to microbial enzymatic factories called nonribosomal
peptide synthetases (NRPSs). NRPSs use contiguous protein domains to incorporate and
assemble simple substrates into complex products in an assembly line fashion. The products
are often valuable therapeutics, including antibiotics (bacitracin), antitumor agents
(bleomycin), and immunosuppressants (rapamycin), but others confer virulence to pathogens
(E. coli, V. cholerae, Y. pestis). NRPSs are the focus of much interest because engineering
them to incorporate different substrates could produce novel pharmaceuticals. However, like
assembly lines in factories, NRPSs are not static, and their domains interact transiently in a
dynamic architecture. Thus, understanding the molecular mechanisms of NRPSs, and
potentially engineering them, is tantamount to solving a dynamic, multi-dimensional puzzle.
Notably, it is unknown how substrates interact with some domains, and how these interactions,
in turn, promote communication between several partner domains, which is the situation we
described above for proteins. We found that structural dynamics within domains respond to
substrates to promote interactions between domains and that they couple remote binding sites
and enzymatic active sites. That is, dynamics contain keys to understanding both substrate
recognition and remote communication. This proposal aims to provide a molecular description of
the dynamics within critical NRPS domains and reveal its function in substrate and partner
domain recognition. We will use nuclear magnetic resonance, which can describe
experimentally dynamics at the atomic-level, to describe dynamic responses when domains
interact with each other, and with substrates as they do during synthesis. The studies are
supplemented with functional assays, computational methods, and crystallography, and will
answer longstanding questions about protein communication, enzyme mechanisms, and remote
communication within proteins. The results will provide a basis to engineer exogenous substrate
recognition into NRPSs, a condition for producing new pharmaceuticals through NRPS
reprogramming.

## Key facts

- **NIH application ID:** 10387089
- **Project number:** 3R01GM104257-07S1
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Dominique Pascal Frueh
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $104,280
- **Award type:** 3
- **Project period:** 2013-06-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10387089

## Citation

> US National Institutes of Health, RePORTER application 10387089, Dynamic allosteric communication within nonribosomal peptide synthetase cyclization domains (3R01GM104257-07S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10387089. Licensed CC0.

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